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The Effect of Different Levels and Sources of Auxin and Cytokinin to Callus Formation on SoybeanAnther Culture

Pengaruh Berbagai Takaran dan Sumber Auksin dan Sitokinin terhadap Pembentukan Kalus padaKultur Antera Kedelai

Z. Zulkarnain

Department of Agroecotechnology, Agricultural Faculty, University of Jambi

ABSTRACT

This investigation was aimed at studying the effect of types and concentrations of auxins and cytokinins on thegrowth and development of anthers of two soybean cultivars, Merubetiri and Wilis cultured in vitro. The trial was conductedat the Plant Biotechnology Laboratory, Agricultural Faculty, University of Jambi. Anthers were cultured on MS solidmedium provided with IAA, 2,4-D or NAA as auxin source in combination with BAP or kinetin as cytokinin source. Eachgrowth regulators was tested at 0, 5, 10, 15 and 20 µM. The experiment was placed in a completely randomized design withfive replicates. Each replicates consisted of 8 to 10 anthers obtained from the same floral bud. Cultures were placed in a lightintensity of 50 µmol m-2.s-1 and 16-hour photoperiod at 25±1 oC. Observation was done weekly for 8 weeks of culture.Results indicated that response showed by anthers cultured on medium supplemented with 2,4-D+BAP, IAA+BAP andNAA+BAP, in the form of callus proliferation, occurred within 5-18 days of culture initiation. Meanwhile, with 2,4-D+kinetin,IAA+kinetin and NAA+kinetin, callus proliferation took place within 4-16 days of culture initiation. Callus formation waspreceded by a swollen on the surface of anthers, followed by changing in color from light green to brownish. Following this,anther wall turned into amorphous shape, before it was finally covered by a white, cream or light green callus mass. Initially,the callus showed friable or compact structure, but following two weeks of proliferation all callus showed compact structure.Among growth regulators tested, combination involving 2,4-D produced more callus than other combinations. In addition,of the two cultivars tested, Merubetiri showed better response compared to Wilis.

Keywords: growth regulators, anther culture, in vitro culture, soybean, Glycine max

ABSTRAK

Penelitian ini bertujuan untuk mengetahui pengaruh berbagai jenis dan konsentrasi zat pengatur tumbuh auksindan sitokinin terhadap pertumbuhan dan perkembangan antera dua kultivar kedelai, Merubetiri dan Wilis, pada kulturin vitro. Penelitian dilaksanakan di Laboratorium Bioteknologi Tanaman, Fakultas Pertanian Universitas Jambi.Antera yang dari dua kultivar kedelai yang diuji dikulturkan pada medium MS padat yang dilengkapi dengan IAA, 2,4-D atau NAA sebagai sumber auksin yang dikombinasikan dengan BAP atau kinetin sebagai sumber sitokinin. Masing-masing zat pengatur tumbuh diberikan pada konsentrasi 0, 5, 10, 15 and 20 µM. Percobaan ini menggunakan rancanganacak lengkap dengan lima ulangan. Setiap ulangan terdiri atas 8-10 antera yang berasal dari kuncup bunga yangsama. Kultur dipelihara di dalam ruangan dengan intensitas cahaya lebih-kurang 50 µmol m-2.s-1 dan fotoperiodesitas16 jam per hari pada suhu 25± 1 oC. Pengamatan dilakukan setiap minggu selama 8 minggu. Hasil penelitian menunjukkanbahwa respons yang diperlihatkan oleh antera yang dikulturkan pada medium yang dilengkapi dengan 2,4-D+BAP,IAA+BAP dan NAA+BAP adalah berupa proliferasi kalus, yang berlangsung dalam waktu 4-16 minggu setelah inisiasikultur. Pembentukan kalus didahului oleh timbulnya pembengkakan pada permukaan antera, diikuti oleh perubahanwarna dari hijau muda menjadi kecoklatan. Selanjutnya, dinding antera berubah bentuk menjadi tidak beraturan,sebelum akhirnya diselimuti oleh massa kalus berwarna putih, krem atau hijau muda. Pada awal perkembangannya,kalus memperlihatkan struktur yang remah dan sebagian kompak, namun setelah dua minggu berproliferasi seluruhkalus memperlihatkan struktur yang kompak. Di antara zat pengatur tumbuh yang diuji, kombinasi yang melibatkan2,4-D menghasilkan kalus yang lebih banyak dibandingkan kombinasi yang lain. Di samping itu, dari dua kultivaryang diuji, Merubetiri memperlihatkan respons yang lebih baik dibandingkan Wilis.

Kata kunci: zat pengatur tumbuh, kultur antera, kultur in vitro, kedelai, Glycine maX

*Penulis korespondensi, e-mail: dr.zulkarnain@yahoo.com

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INTRODUCTION

Haploid technology offers more advantages thanconventional method. By this technique, homozigousplants could be developed within one generation, whileconventional method requires selection processesinvolving 5-6 generations to produce homozigous plants(Taji et al., 2002). Haploid individuals also provide anexcellent example when studying induced mutagenesis,where recessive traits can be easily detected (Seguí-Simarroand Nuez, 2008b). A number of recessive traits such astolerance to unfavourable conditions, such as drought,cold, heavy metals or low nutrients, are amongst recessivetraits that can be detected promptly in haploid plants(Tapingkae et al., 2012). Further, problems associated withoutcrossing and self incompatibility can also be overcomeby the use of haploid technology.

Haploid plants can be obtained through antherculture. Anthers obtained from young floral buds may beaseptically isolated and used as plant materials in tissueculture. Immature microspores within these anthers maybe induced to grow and give rise to complete plants underfavourable conditions. Since microspores are haploid, plantsregenerated from microspore cells will also be haploid.Haploid plants have no homologous set with which to pair;the normal pairing of chromosomes during meiosis cannottake place. Consequently, sterile plants or plants with non-functional male sexual organs are produced. When thechromosome complements are artificially doubled, e.g. usingantimitotic chemicals such as colchisin or oryzalin, theregenerated plants will be doubled-haploid. As withhomozigous haploids, the regenerated doubled-haploidindividuals are also homozigous. The difference is that thedoubled-haploid plants are fertile, and therefore can bepropagated sexually.

Haploid plants regenerated from tissue system culturewere first reported by Guha and Maheshwari (1964) onDatura inoxia and followed by Nitsch and Nitsch (1969)on tobacco (Nicotiana tabacum). Since then, success hasalso been reported on various crops such as Lupinus spp.(Bayliss et al., 2004), Glycine max, Vigna unguiculata ,Psophocarpus tetragonolobus, Albizzia lebbeck andPeltophorum pterocarpum (Crosser et al., 2006), Brassicasp. (Alam et al., 2009), Oryza sativa (Khatun et al., 2012)and Populus x beijingensis (Li et al., 2013).

The application of plant growth regulators to culturemedium is necessary for the successful induction ofmicrospore embryogenesis. Auxin and cytokinin are thetwo most extensively used growth regulators in the antherculture of a wide range of plant species. Auxin such as 2,4-dichlorophenoxyacetic acid (2,4-D) was usually applied inthe anther culture of Triticum aestivum (El-Hennawy et al.,2011) and Brassica napus (Ardebili et al., 2011). In addition,vigorous green plantlets were regenerated from microsporeculture of Hordeum vulgare in the presence of auxins suchas indoleacetic acid (IAA) or naphthaleneacetic acid (NAA)(Castillo et al., 2000).

Kinetin and benzylamino purine (BAP) are twocytokinins that promote shoot regeneration from withincallus derived from Oryza sativa (Bishnoi et al., 2000) andembryogenesis in Cucumis sativus L. (Hamidvand et al.,2013) anther cultures. In addition, Rukmini et al. (2013)claimed that the combination of kinetin+BAP+NAA provedto be optimal for callus induction and green plantregeneration in two Indica rice (Oryza sativa L.) hybrids -Ajay and Rajalaxmi.

This study was aimed at investigating the effect ofdifferent levels and sources of auxins and cytokinins onthe growth and development of soybean cv. Merubetiriand Wilis anthers cultured in vitro.

METHODOLOGY

Stock plant preparationThe stock plants used in this investigation were two

soybean cultivars, i.e. Merubetiri and Wilis grown inglasshouse. Seeds were germinated and grown in blackpolyethylene plastic bags. Care of plants followed generalcultivation technique including watering, pests anddiseases controlling, weeding and fertilizer application toobtain healthy growth. Seed germination were carried outevery 3-4 weeks to ensure adequate availability of floralbuds during investigation.

Explant sourceAnthers obtained from 2.5-3.5 mm long floral buds

were used as planting materials in this study. Floral budsfrom glasshouse-grown plants were isolated and dipped in70% alcohol for 10 seconds. Following from this, sepal andpetal were carefully removed and anthers were separatedfrom filaments prior to culture in prepared media.

Culture mediumIn this study we used solid MS (Murashige and

Skoog, 1962) basal medium supplemented with vitaminsand 3% sucrose, and the pH of the medium was adjusted to5.6 0.2. Prior to sterilizaton in autoclave at 1.1 kg cm-1 (103kPa) and temperature of 121 oC for 20 minutes, 8 g BactoBitek agar was added to the medium.

Variables testedGrowth regulators tested were IAA, NAA or 2,4-D

(each at 0, 5, 10, 15 and 20 µM) as auxin sources, incombination with BAP or kinetin (each at 0, 5, 10, 15 and 20µM) as cytokinin sources. Therefore, there were 25treatment combinations, each repeated 4 times resulted in100 experimental units in a Simple Completely RandomizedDesign. Each experimental unit consisted of 4 culture flaskscontaining one immature leaf segment explants each.

Culture maintenance and observationCultured anthers were kept in a growth room with

temperature of 25 1oC. Photoperiod was adjusted to 16

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hours per day and light intensity approximately50 µmol m-2.s-1 obtained from fluorescent lamp.

Anthers growth and development were observed dailyfor eight weeks of culture period. Variables observed werethe percentage of explants forming callus, time to callusformation following culture initiation, and thecharacteristics of callus (colour and structure).

Quantitative data were analyzed by ranking of meanmethod and presented in the form of tables, whereasqualitatif data were presented visually in the form of picture/photograph.

RESULTS AND DISCUSSION

Anthers from the two soybean cultivars (Merubetiriand Willis) cultured on solid MS medium supplementedwith IAA, NAA or 2,4-D in combination with BAP or kinetinshowed callus formation within 5-18 days after culture.Callus proliferation was started by swollen on the antherssurface followed by changes in their colour from light greeninto brownish. Following from this, the anther wall showedamorphous structure, and finally the whole anthers werecovered by callus mass. Meanwhile, on non callus formingexplants the colour of anthers turning into white or brownand show no further development.

The percentage of explant forming callusMedium supplemented with IAA + BAP

On Wilis, of all tested combination, callus formationwas only found on anther cultured on mediumsupplemented with 15µM IAA+10 µM BAP. Whereas othercombinations of plant growth regulators did not show anyeffect on explant development. All cultured explants turningwhite and /or brown indicating that they are all dead withoutshowing any callus proliferation.

The same condition was also observed on Merubetiri,in which callus formation occurred only on explants culturedon medium supplemented with 15µM IAA+10 µM BAP, 20µM IAA+5 µM BAP and 20µM IAA+15 µM BAP. Similaras Wilis, non callus forming explants turned into white orbrown and finally died.

Medium supplemented with IAA+kinetinSimilar to IAA+BAP treatment, neither anthers

isolated from Wilis nor Merubetiri cultured on mediumsupplemented with IAA+kinetin showed satisfatoryresponses. On Wilis, callus was only formed on mediumsupplemented with 20 µM IAA+10 µM kinetin and mediawith 20 µM IAA+20 µM kinetin. Similar response was alsoshown by Merubetiri. Callus proliferation only occurredon anthers cultured on media supplemented with 5 µMIAA+15 µM kinetin and 10 µM IAA+20 µM kinetin.Meanwhile, anthers cultured on other media turned whiteor brown without showing any callus proliferation.

Medium supplemented with 2,4-D+BAPOn Wilis, callus formation occurred on explants

cultured on medium supplemented with 5 µM 2,4-D+15 µMBAP, 10 µM 2,4-D without BAP dan 20 µM 2,4-D withoutBAP. Meanwhile, on Merubetiri, callus were observed onanthers cultured on medium with 20 µM BAP without 2,4-D, 5 µM 2,4-D+10 µM BAP, 15 µM 2,4-D+10 µM BAP, 15µM 2,4-D+15 µM BAP, 20 µM 2,4-D+without BAP and 20µM 2,4-D+10 µM BAP (Table 1). Explants cultured on othermedia did not show any response, but their colour turnedwhite and/or brownish and died eventualy.

Medium supplemented with 2,4-D + kinetinThe presence of 2,4-D in culture medium was found

to be important for callus formation on soybean anthers.This was indicated by the proliferation of callus mass onnearly all treatments except kinetin without 2,4-D, 5 µM2,4-D without kinetin 5 µM 2,4-D+15 µM kinetin, 5 µM 2,4-D+20 µM kinetin, 10 µM 2,4-D without kinetin, 15 µM 2,4-D without kinetin, 15 µM 2,4-D+15 µM kinetin, and 15 µM2,4-D+20 µM kinetin. Meanwhile on Merubetiri, callusproliferation occured on anthers cultured on a number of2,4-D+kinetin combination, but medium without 2,4-D, 5µM 2,4-D without kinetin, 15 µM 2,4-D+5 µM kinetin, 15µM 2,4-D+20 µM kinetin, and 20 µM 2,4-D+5 µM kinetin.

Data on callus formation from anthers of the twosoybean cultivars cultured on media supplemented with2,4-D+kinetin are presented in Table 2.

Table 1. The percentage of soybean anthers forming callus when cultured on medium supplemented with 2,4-D + BAP

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Table 2. The percentage of Wilis and Merubetiri anthers forming callus following culture on medium supplemented with2,4-D+kinetin.

Medium supplemented with NAA+BAPAnthers isolated from Wilis showed response when

cultured on media supplemented with 10 µM BAP alone,10 µM NAA+5 µM BAP, 15 µM NAA+15 µM BAP, 20 µMNAA alone, and 20 µM NAA+20 µM BAP. On the otherhand, anthers obtained from Merubetiri resulted in responsewhen exposed to culture media supplemented with 10 µMBAP without NAA, BAP 20 µM without NAA, 5 µM NAA

without BAP, 5 µM NAA+10 µM BAP, 10 µM NAA withoutBAP, 10 µM NAA+10 µM BAP, 10 µM NAA+15 µM BAP,10 µM NAA+20 µM BAP, 15 µM NAA+15 µM BAP, 15 µMNAA+20 µM BAP, 20 µM NAA without BAP, 20 µMNAA+5 µM BAP, and 20 µM NAA+20 µM BAP.

Data presented on Table 3 shows the percentage ofanthers from the two soybean cultivars showing responsewhen cultured on media supplemented with NAA+BAP.

Table 3. The percentage of Wilis and Merubetiri anthers forming callus following culture on medium supplemented withNAA+BAP

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Medium supplemented with NAA+kinetinWhen cultured on medium supplemented with

NAA+kinetin, callus proliferation was found on Wilis antherexposed to 10 µM NAA+5 µM BAP, 15 µM NAA alone, 15µM NAA+5 µM BAP, 20 µM NAA+5 µM BAP, and 20 µMNAA+10 µM BAP. Meanwhile, on Merubetiri responsewas shown by anthers cultured on medium with 5 µM

NAA+10 µM BAP, 5 µM NAA+20 µM BAP, 10 µM NAA+5µM BAP, 10 µM NAA+10 µM BAP, 10 µM NAA+15 µMBAP, 10 µM NAA+20 µM BAP, 15 µM NAA without BAP,15 µM NAA+15 µM BAP, 15 µM NAA+20 µM BAP, 20 µMNAA+5 µM BAP, and 20 µM NAA+10 µM BAP.

Table 4 shows the percentage of anthers formingcallus from the two soybean cultivars when cultured onmedia supplemented with NAA+kinetin.

Table 4. The percentage of Wilis and Merubetiri anthers forming callus following culture on medium supplemented withNAA+kinetin.

Table 5. Time to callus initiation on anthers of soybean cv. Wilis and cv. Merubetiri cultured on medium supplemented withBAP plus different sources of auxin.

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The time to callus proliferation after culture initiationData collected during the investigation showed that

callus proliferation on the surface of anthers cultured onmedium supplemented with BAP occurred within 5 to 18days after culture initiation (Table 5). Meanwhile, on mediumsupplemented with kinetin callus proliferation was foundwithin 4 to 16 days following culture initiation (Table 6).

The colour of callusIn general, medium supplemented with BAP+IAA or

BAP+NAA produced callus that was initially greenytransparent in colour. On the other hand, white callus wasinitiated on anthers cultured on medium enriched withBAP+2,4-D which then turned creamy transparent (Figure1). On medium with IAA as auxin source, the greenytransparent callus gradually turned light green before

eventually turning dark green (Figure 2A,B). Meanwhile,on medium supplemented with NAA the initially greenytransparent callus turned whitish green or light greenfollowing 2 weeks of proliferation (Figure 3).

In Wilis, the changes in colour was mostly on callusproliferated from anthers cultured on medium with NAA,whereas in Merubetiri the changes in colour was foundonly on callus formed on anthers cultured on medium with20 µM NAA+20 µM BAP. In addition to whitish green,some callus regenerated on Merubetiri anthers also turnedinto dark green, especially those cultured on medium with20 µM BAP alone, 15 µM NAA+20 µM BAP and 20 µMNAA+20 µM BAP, or light green on medium with 15 µMNAA+20 µM BAP, 10 µM NAA+20 µM BAP and 20 µMNAA+20 µM BAP.

Table 6. Time to callus initiation on anthers of soybean cv. Wilis and cv. Merubetiri cultured on medium supplemented withkinetin plus different sources of auxin.

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Figure 1. White callus proliferated on anthers cultured onmedium supplemented with 2,4-D + BAP.

Figure 2. Light green (A) and dark green (B) callus proliferatedon anthers cultured on medium supplemented withIAA + BAP.

Figure 3. Whitish green callus proliferated on anthers culturedon medium supplemented with NAA + BAP.

Figure 4. White, creamy yellow and greenish callus proliferatedon anthers cultured on medium supplemented withkinetin as cytokinin source in combination with 2,4-D (A), IAA (B) and NAA (C) as auxin sources (2weeks after culture initiation).

Four weeks after culture initiation, the light greencallus started browning on the side exposed to culturemedium. On the other hand, white to creamy yellow callustook longer time to brown, approximately 10 weeks afterinitiation. Meanwhile, callus proliferated from anthers ofthe two soybean cultivars cultured on mediumsupplemented with kinetin in combination with 2,4-D, IAAor NAA relatively similar in colour. In general, in spite ofauxin sources, the colour of callus regenerated on mediumwith kinetin as cytokinin source was white transparent tocreamy yellow (Figure 4).

Callus structureAt the begining of its proliferation, all callus

regenerated from anthers cultured on all auxin and cytokinincombinations showed the same structure, ie compact tofriable. Following 4 weeks of proliferation, however, allthose callus turned into compact structure (Figure 5).

DiscussionAll regenerated callus proliferated from within

anthers following the broken of anther wall. This indicatesthat there was possibility that those callus originated frommicrospores within the anthers. Therefore, the ploidy levelof regenerated callus presumably the same as the ploidylevel of the microspores, ie haploid. This assumption,however, need to be proven by examining and countingthe number of the chromosome under microscope. Anotherway to prove the callus was haploid was to induceembryogenesis from within callus mass. It was hoped thatembryoids raised from callus were haploid and grew intocomplete haploid plants.

This investigation revealed that the involvement ofplant growth regulators, especially auxin, in culture mediumsignificantly affected callus formation. Among testedauxins, 2,4-D was more effective than IAA or NAA indicatedby greater number of explant forming callus. The use of2,4-D as an auxin source for callus induction in legumeshave also been reported in previous works in which 1-2mg.L-1 2,4-D was the most effective (Kiran et al., 2005;Kaviraj et al., 2006; Kumari et al., 2006; Ahlawat et al.,2013). This can be understood since 2,4-D is categorizedas a strong auxin having important role in stimulating theformation of callus under in vitro culture system.

Though the application of BAP or kinetin incombination with 2,4-D, IAA or NAA was important inpromoting callus proliferation, the characteristic of callusregenerated from anthers of the two soybean cultivars,however, was the same. The colour of callus was white togreenish white and compact in structure. In other words,the effect of plant growth regulators showed no correlationto test soybean genotype. Similar effect of growthregulators was also reported by Zulkarnain et al. (2002) inanther culture of Swainsona formosa, a leguminousornamental plant native to Australia.

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Figure 5. The compact structure of callus proliferated on anhersof soybean cv. Merubetiri cultured on medium withBAP (A) and kinetin (B) as cytokinin source (4weeks after proliferation).

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The white colour and compact structure indicatedthat there was a embryogenic capacity of respected callus.This had been proven by a number of investigators, eitheron leguminous or non-leguminous plants. For example, inin vitro culture of Bixa arellana, Sha-Valli-Khan et al. (2002)found that the combination of NAA + BAP resulted inshiny white friable callus, that later turned white andcompact before developing green globular structure. Thedevelopment of such green globular structure indicatedthe early stage of embryogenesis as reported by Sudhersanand Abo-El Nil (2002) and Zulkarnain (2004) in in vitroculture of Swainsona formosa. The formation of white andcompact callus that ended with embryogenesis was alsoreported by Fulzele and Satdive (2003) in tissue culture ofNothapodytes foetida and by Mohajer et al. (2012) inOnobrychis sativa, an important forage legumes.

Meanwhile, of the two tested soybean cultivars, itwas found that Merubetiri was more responsive than Wilisunder in vitro system. The effect of genotype on the invitro culture of legumes had also been reported on Cicerarietinum (Arora and Chawla, 2005); Khan et al. (2011),Phaseolus vulgaris (Arias et al., 2010) and Cyamopsistetragonoloba and C. serrata Mathiyazhagan et al. (2013).In addition, variability in organogenic responses amonggenotypes was commonplace in numerous grain legumesincluding Lathyrus sativus (L.) (Ochatt et al., 2007) andVigna subterranea (Koné et al., 2007; Koné et al., 2013).

It is obvious that the response of explants during invitro cul-ture is dependent upon the genotype of the donorplants. It is not only different species, but also differentcultivars within species and, even individuals of the samecultivar that may show differences in embryogenicresponses (Seguí-Simarro and Nuez, 2008a). Although thebasis of genetic control is not understood yet, it was clearthat genetic factors interacted with other factors to controlthe direction of explant development under in vitro culturesystem.

Based on reports on previous investigations, theregeneration of white and compact callus in this studyimplies a great opportunity to obtain haploid plants viaembryogenesis from within callus mass that presumablyconsisted of haploid cells. By making some modificationson a number of environmental factors, particularly mediumcomposition, it is hoped that the production of haploidsoybeans via in vitro culture system could be realized, atleast on Merubetiri that was more responsive than Wilis.

CONCLUSION

Based on the results obtain in this investigation, itcan be concluded that:1. The presence of plant growth regulators, particularly

IAA, NAA or 2,4-D and BAP or kinetin, in culturemedium was crucial in promoting callus formation on

anther culture of soybean, either on Merubetiri or Wiliscultivars. However, Merubetiri was found to producebetter in vitro response compared to Wilis.

2. Among tested auxins and cytokinins, the combinationof 20 µM 2,4-D with 10 µM to 20 µM kinetin showedbetter results than other combinations.

3. Based on this reported investigation, future studyshould focus on in vitro culture of Merubetiri alongwith the use of 2,4-D and kinetin.

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